傅里叶红外光谱技术在法医学脑挫裂伤鉴定中的应用展望
左莹莹1, 李健2,*
1.北京市公安局石景山分局,北京100043
2.北京警察学院,北京102202
* 通讯作者简介:李健,男,河南商丘人,硕士,主任法医师,研究方向为法医病理学。E-mail: lijian199307fy@163.com

第一作者简介:左莹莹,女,四川眉山人,硕士,主检法医师,研究方向为法医病理学和法医物证学。E-mail: 1958yybb@163.com

摘要

脑挫裂伤是常见的一类颅脑损伤,单纯的组织病理学技术可能难以早期发现及精确诊断。傅里叶变换红外面扫描成像技术(FTIR-mapping)是病理学研究领域的新技术,因其结合了傅里叶变换红外光谱、红外显微镜技术以及面扫描成像技术的特点,具有测定方法简便、对样本无损、灵敏度高、准确、直观等优点,突破了传统技术的局限,能检测出发生了物质数量、结构以及构象变化但缺乏明显形态学变化的脑挫裂伤,从而实现早期精准地诊断脑挫裂伤。FTIR-mapping虽已能绘制某些损伤组织的红外光谱病理图像,但其在法医病理学中的价值有待全面深入开发。本文综述了脑挫裂伤的法医病理学研究进展以及傅里叶变换红外面扫描成像技术在法医病理学中的应用。

关键词: 法医病理学; 脑挫裂伤; 傅里叶变换红外光谱; 红外显微镜技术; 面扫描成像技术
中图分类号:DF795.1 文献标志码:A 文章编号:1008-3650(2020)02-0183-05
Insight on Applications of Fourier Transform Infrared Spectroscopy into Forensic Identification of Cerebral Contusion or Laceration
ZUO Yingying1, LI Jian2,*
1. Shijingshan Branch of Beijing Public Security Bureau, Beijing 100043, China
2. Beijing Police College, Beijing 102202, China
Abstract

Cerebral contusion/laceration is one commonly-seen craniocerebral injury. Single histopathological techniques are difficult to early detect or accurately diagnose cerebral contusion/laceration. Fourier transform infrared spectrum mapping (FTIR-mapping), a new approach for pathology, integrates the specifics of Fourier transform infrared spectroscopy, infrared microscopy and mapping, therefore making it assume the advantages of simple measuring, no damaging to sample, high sensitivity, accuracy and convenience for observation. FTIR-mapping breaks through the limitations of traditional technology, with the ability to detect the cerebral contusion/laceration that has changed in quantity, structure and conformation yet lacked obvious morphological alterations, thus realizing the early and accurate diagnosis of cerebral contusion/laceration. Although FTIR-mapping has presently been able to depict the infrared spectral pathologic images of some damaged tissues, its potential in forensic pathology is still expecting to be thoroughly developed. This article reviews the progress of forensic pathological studies on cerebral contusion/laceration, with inclusion of the applications of FTIR-mapping into forensic pathology.

Keyword: forensic pathology; cerebral contusion/laceration; Fourier transform infrared spectrum (FTIR); infrared microscopy; mapping
1 脑挫裂伤的法医病理学研究进展
1.1 脑挫裂伤的含义及研究意义

脑挫裂伤, 是外力作用于头部造成的原发性脑组织器质性损伤。脑挫伤和脑裂伤统称为脑挫裂伤, 前者损伤病灶大小不等, 小者如点状出血, 大者可聚合成片, 灶中央以出血为主, 四周可见脑组织各种成分坏死, 软脑膜尚完整; 后者为软脑膜、血管和脑组织同时有破裂, 伴蛛网膜下腔出血[1]

颅脑损伤患者的死亡率与致残率高[2], 已成为全球重要公共卫生问题之一。同时, 颅脑损伤也是法医实际工作中最重要的损伤和最常见的死因之一[3]。脑挫裂伤是常见的一类颅脑损伤, 研究脑挫裂伤能为诊断和治疗该损伤提供理论依据, 为判断案件性质和定罪量刑提供科学依据, 对临床医学以及法医学都有重要的指导意义。

1.2 脑挫裂伤的研究方法进展

脑挫裂伤研究方法是以传统的镜检为基础, 随着现代免疫学、分子生物学等学科的理论和技术的迅速发展, 对脑挫裂伤的研究也进一步深入到亚细胞、分子和基因水平。近几年, 关于脑挫裂伤的研究动态主要集中在神经元细胞、各类神经胶质细胞(尤其是星形胶质细胞)及其分泌的细胞因子、生物酶和各类炎性介质等研究对象上[4, 5]

有些研究者采用免疫组织化学技术研究脑挫裂伤, 有的研究发现人脑挫伤后的胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)阳性细胞的面积和灰度随时间推移呈先增加后下降的趋势[6]; 还有研究发现大鼠脑挫伤后原癌基因c-fos的表达产物Fos蛋白和神经生长因子受体(nerve growth factor receptor, NGFR)在损伤局部的阳性染色分别在伤后早期及随后较晚时段里出现, 前者表达时间短暂, 后者则持续时间较长, 都有一定的规律性[7]。还有研究者采用TUNEL和Feulgen's DNA染色以及图像分析技术在分子水平上研究大鼠脑挫伤, 发现随着损伤时间延长, 神经细胞DNA片段化程度逐渐增强、含量逐渐降低[8]。酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)也被用来研究脑挫裂伤, 结果显示脑挫裂伤后3 d和7 d, 伤者血浆水平内皮素-1(endothelin-1, ET-1)和降钙素基因相关肽(calcitonin gene-related peptide, CGRP)的含量均高于对照组[9]

1.3 法医学传统方法研究脑挫裂伤的局限

目前, 脑挫裂伤的法医学鉴定主要依赖于检测上述相关分子物质的含量、结构改变的组织病理学技术, 包括HE染色、银染法、免疫组织化学法等。然而, 这些方法都存在一定的局限性[10, 11]。如银染法不能诊断死亡过程迅速的脑挫裂伤, β -淀粉样前体蛋白(β -amyloid precursor protein, β -APP)免疫染色阳性并非脑挫裂伤独有的特征性反应, 在伴有颅内压增高的缺血、缺氧性脑损伤、疝性脑损伤中均可出现β -APP阳性表达的现象。其他生物标记, 如神经丝(neurofilament, NF), 神经丝致密化(neurofilament compaction, NFC)、微管相关蛋白(microtubule associated proteins, MAP)等也存在类似的缺陷[12, 13, 14]。所以, 单纯的组织病理学技术可能难以早期发现及精确诊断脑挫裂伤。

2 傅里叶变换红外面扫描成像技术的原理和应用
2.1 傅里叶变换红外光谱的原理和应用

傅里叶变换红外光谱(Fourier transform infrared spectrum, FTIR)是一种能够在保留样本原始特性的情况下, 同时检测样本中多种大分子物质成分及结构情况的技术。由于其具有测定方法简便、对样本无损、灵敏度高、可同时测定多种成分等特点, 广泛应用于医学、药学、食品科学、化工、地质、环境监测等众多领域[15, 16]。FTIR主要研究分子中以化学键联结的原子之间的振动光谱和分子的转动光谱。FTIR及其相关技术飞速发展, 不仅广泛用于研究核酸、蛋白质等生物大分子的结构, 而且已经用于研究诸如细胞、组织等更为复杂的体系。由于各种细胞的红外振动信号各不相同, 因此可根据这些差异来区分不同的组织细胞[17, 18]。核酸(DNA和RNA)、蛋白质、双层磷脂膜等是人体组织细胞中最基本的聚合物, 这些聚合物分子的振动光谱组成了细胞的红外光谱[19, 20]。它反映了核酸、蛋白质、糖蛋白等分子和生物膜在细胞内的含量、构型、构象及其所发生的变化。蛋白质、脂类、碳水化合物和核酸等构成组织和细胞的主要物质在组织和细胞变性、坏死过程中, 会在数量、结构和构象上发生明显的病理改变, 但是传统的形态学方法往往无法在早期检测到这些变化[10, 21]。理论上讲, 缺乏明显形态学变化的脑挫裂伤, 只要在物质数量、结构或构象上发生了变化, 虽然还不能通过传统病理形态学或其他方法检测出来, 但都可被FTIR检测到[22, 23, 24]。这使得利用FTIR在早期发现脑挫裂伤成为可能。因而, 运用FTIR理论研究病理变化被视为一种新的学说或理论, 称作“ 红外光谱病理学” [25]

2.2 红外面扫描成像技术的原理及法医病理学应用

FTIR与红外显微镜技术的联用实现了将显微观察与红外光谱测定的有机结合, 可以直观地区分样本各主要结构或在一次检测中观察多种成分的分布情况, 做到所测即所见, 并且还可与其他技术结果形成良好的对应关系[26, 27]。此外, 不断开发出的配套计算机软件能够自动进行光谱数据的处理, 使光谱分析更为快速和准确。

红外图像分析技术是在傅里叶变换红外显微镜技术的基础上发展起来的微区分析技术, 显微红外面扫描成像技术(FTIR-mapping)可通过红外图像的不同色差来区分不同的组织细胞, 能直观显示组织病理改变。红外显微镜技术的灵敏度明显高于传统方法, 使得FTIR-mapping表现出很多优势, 如分辨率为25 μ m× 25 μ m, 可以将一张组织样品切片分为60 000个单独检测区域, 检测过程中采集这60 000个区域的红外光谱信息, 这60 000个像素信息反映组织细胞的病理变化, 经过系统分析, 计算积分面积, 便可获得样品上各感兴趣区域的某一组分的含量分布图, 再通过拼接每种组分的红外光谱信息, 就能合成整张光谱图像。更重要的是, FTIR-mapping图像由计算机自动生成, 能将病变部位用特定的颜色突出呈现, 消除了个体主观判断的影响, 避免传统人工阅片引发的错误, 结果更加准确、直观。因此, FTIR-mapping代表了一种病理形态学研究的新方法, 又被称为“ 图像分子化学” [28, 29, 30]。FTIR-mapping的这些优点为精确诊断脑挫裂伤提供了理论支撑。FTIR-mapping现已能够绘制某些损伤组织的红外光谱病理图像[31, 32]。相对可见光谱而言, FTIR-mapping将人类视野拓展至红外光谱, 从分子学水平研究病理变化, 拓展了传统病理形态学的研究领域, 以期解决现有技术手段不能解决的问题。

3 红外光谱技术在法医病理学脑挫裂伤研究中的应用展望

近些年来, 红外光谱的应用逐渐拓展到法医病理学领域。FTIR被用于检测大鼠外伤性癫痫脑组织中的α -螺旋结构跨膜蛋白[33]、人心肌梗死组织[34]、人脑转移瘤[35]和小鼠脑肿瘤中的胶原成分[36]、大鼠外伤性脑损伤中受损轴突的蛋白质[37]、大鼠外伤性轴索损伤后不同时间点胼胝体中的大分子物质[38, 39], 从而揭示外伤性癫痫的病理生理过程[33], 鉴别早期心肌缺血和陈旧性心肌梗死[34], 界定脑转移瘤的肿瘤范围[35], 预测脑转移瘤的原发肿瘤[35], 区分正常组织、实体瘤和弥漫性肿瘤[36], 诊断轴索损伤[37], 鉴别外伤性轴索损伤后不同时间点的正常脑白质损伤[38], 评估外伤性轴索损伤的损伤时间间隔[39]。有研究者综述了FTIR在法医学的应用, 例如人体损伤检验、死亡时间推断、死亡原因判定、药毒物分析等[40]。还有研究者通过FTIR检测弥漫性轴索损伤过程中蛋白质的病理变化, 并以明显色差图像展示其病理形态学变化特征, 绘制红外光谱图像, 发现红外图像所示同银染以及β -APP免疫组化染色证实的损伤区域分布一致, 实现了FTIR对弥漫性轴索损伤的病理形态学诊断[41], 这为红外光谱检测脑挫裂伤提供了参考。

回顾前人研究, 还未发现利用红外光谱技术从法医病理学角度针对脑挫裂伤进行的研究。结合红外光谱技术定性[42]、定量[43]、定位以及定时[44]的特点, 可运用该技术诊断脑挫裂伤, 划定损伤范围, 推断损伤时间; 同时也会对于法医学其他方面的研究和应用提供全新的思路和方法。因此, 这将有助于准确及时地诊断脑挫裂伤, 分析其在法医学死因构成中的作用, 对判断案件性质、保障刑事诉讼的顺利进行都将发挥一定的作用。

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